Several publications are referenced in this application to more fully describe the state of the art to which this invention pertains. The entire disclosure of each such publication is incorporated by reference herein.
Bacteria regulate gene expression in response to changes in cell population density by a process called quorum sensing. Specifically, quorum sensing bacteria release and detect chemical signals called autoinducers. Bacteria respond to the accumulation of a minimal threshold stimulatory concentration of autoinducer. Detection of autoinducers enables bacteria to distinguish between low and high cell population density, and to control target gene expression in response to fluctuations in cell number. Quorum sensing bacteria regulate processes that require the cooperation of a number of bacterial cells in order to be effective, and the individuals in the group profit from the activity of the entire assembly (Bassler, 1999; Fuqua et al., 1996; Kleerebezem et al., 1997; Lazazzera and Grossman, 1998; Miller and Bassler, 2001; Schauder and Bassler, 2001; de Kievit and Iglewski, 2000). These processes include bioluminescence, virulence, antibiotic production, sporulation and biofilm formation. Quorum sensing therefore allows a population of bacteria to coordinate behavior, and thus take on the characteristics of multi-cellular organisms.
In general, quorum sensing is controlled by acyl-homoserine lactone autoinducers in Gram-negative bacteria and by modified oligopeptide autoinducers in Gram-positive bacteria (Miller and Bassler, 2001; Schauder et al., 2001; Lazazzera and Grossman, 1998; de Kievit and Iglewski, 2000). Gram-negative quorum sensing circuits typically resemble the canonical circuit of Vibrio fischeri. Specifically, the acyl-HSL autoinducer synthase is similar to the V. fischeri LuxI enzyme, and a transcriptional activator similar to the V. fischeri LuxR protein is responsible for autoinducer recognition and target gene activation (Engebrecht et al., 1983; Engebrecht and Silverman, 1984; 1987; Miller and Bassler, 2001; de Kievit and Iglewski, 2000). In Gram-positive bacteria, the oligopeptide autoinducers are synthesized as precursor peptides that are processed, modified and subsequently secreted by ATP Binding Cassette (ABC) type exporters. Gram-positive bacteria detect and respond to oligopeptide autoinducers via two-component phosphorylation cascades (Kleerebezem et al., 1997; Lazazzera and Grossman, 1998; Miller and Bassler, 2001). Both acyl-HSL and oligopeptide autoinducers are highly specific to the species that produce them, as autoinducers produced by one species usually do not influence expression of genes in other species. It is remarkable that such signaling specificity exists in both types of quorum sensing circuits, given the similarity in the members of each class of signal.
Unlike all other quorum sensing bacteria, Vibrio harveyi, a bioluminescent Gram-negative bacterium, uses a novel regulatory circuit to control quorum sensing. Specifically, V. harveyi controls density dependent expression of the luciferase genes using a hybrid quorum sensing circuit, with components common to both Gram-negative and Gram-positive quorum sensing systems (Bassler, 1999). Like other Gram-negative quorum sensing bacteria, V. harveyi uses an acyl-homoserine lactone autoinducer (called AI-1) as a signal (Bassler et al., 1993; Cao and Meighen, 1989). However, similar to Gram-positive quorum sensing bacteria, V. harveyi employs a two-component signaling circuit for autoinducer detection and signal transduction (Bassler et al., 1993; Bassler et al., 1994a; b; Freeman and Bassler, 1999a; b; Freeman et al., 2000; Lilley and Bassler, 2000). In addition, a second, novel autoinducer, termed AI-2, also regulates quorum sensing in V. harveyi (Bassler et al., 1994a; Surette and Bassler, 1998; Surette et al., 1999). It is hypothesized that V. harveyi uses AI-1 for intra-species cell-cell communication and AI-2 for inter-species cell-cell signaling (Bassler et al., 1997; Bassler, 1999; Surette et al., 1999). These distinct signals presumably allow V. harveyi, which inhabits multi-species consortia, to vary its gene expression not only in response to changes in total cell number, but also in response to fluctuations in the species composition of the community.
Thus, V. harveyi has two independent density sensing systems (called Signaling Systems 1 and 2), and each is composed of a sensor-autoinducer pair. V. harveyi Signaling System 1 is composed of Sensor 1 and autoinducer 1 (AI-1), and this autoinducer is N-(3-hydroxybutanoyl)-L-homoserine lactone (see Bassler et al., Mol. Microbiol. 9: 773–786, 1993). V. harveyi Signaling System 2 is composed of Sensor 2 and autoinducer 2 (AI-2) (Bassler et al., Mol. Microbiol. 13: 273–286; 1994). Signaling System 1 is a highly specific system proposed to be used for intra-species communication and Signaling System 2 appears to be less species-selective, and is hypothesized to be for inter-species communication (Bassler et al., J. Bacteriol. 179: 4043–4045, 1997). Reporter strains of V. harveyi have been constructed that can produce light exclusively in response to Al-1 or to AI-2 (Bassler et al., 1993, supra; Bassler et al., 1994, supra).
Quorum sensing in V. harveyi, mediated by Signaling Systems 1 and 2, triggers the organisms to bioluminesce at a certain cell density. These same signaling systems, particularly Signaling System 2, are believed to trigger other physiological changes in V. harveyi and other bacteria possessing the same signaling system.
Consistent with a role for AI-2 as a universal signal used for bacterial inter-species communication, over 30 species of Gram-negative and Gram-positive bacteria have now been shown to produce AI-2 (Bassler et al., 1997; Miller and Bassler, 2001; Surette and Bassler, 1998, the disclosures of which are incorporated herein by reference in their entireties). In every case, an AI-2 synthase that is highly homologous to the V. harveyi AI-2 synthase called LuxS is required for AI-2 production (Surette et al., 1999). Recently, the biosynthetic pathway for AI-2 synthesis was described (Schauder and Bassler, 2001; Schauder et al., 2001). AI-2 is produced from S-ribosylhomocysteine (SRH), a product in the S-adenosylmethionine (SAM) utilization pathway. Specifically, LuxS cleaves SRH to form homocysteine and AI-2. Although not confirmed, AI-2 appears to be a furanone with structural similarity to ribose (Schauder et al., 2001). The current evidence suggests that, in contrast to the variable structures of acyl-homoserine lactone and peptide autoinducers, the structures of AI-2 from different species of bacteria are identical. If AI-2 is used for inter-species signalling in natural habitats, a common signal structure could be required for it to be recognized by multiple members of a mixed-species community (Schauder et al., 2001; Schauder and Bassler, 2001).
Although the role of AI-2 is understood in the regulation of bioluminescence in V. harveyi, what function AI-2 plays, if any, in other luxS-containing bacteria is not clear. There are reports showing that AI-2 is involved in regulating type III secretion in E. coli 0157:H7 (Sperandio et al., 1999), protease production in Streptococcus pyogenes (Lyon et al., 2001), hemolysin production in V. vulnificus (Kim et al., 2000), and regulation of the virulence factor VirB in Shigella flexneri (Day and Maurelli, 2001). Genetic experiments established how AI-2 regulates gene expression in Salmonella typhimurium, and show that AI-2 controls the expression of a previously uncharacterized operon encoding an ABC transporter apparatus that appears to function in the uptake of AI-2.
Definitions:
Various terms relating to the biological compounds of the present invention are used throughout the specifications and claims. The terms “substantially the same,” “percent similarity” and “percent identity” are defined in detail below.
The novel signaling factor of the present invention is alternatively referred to herein as “signaling factor”, “signaling compound”, “autoinducer”, and more specifically, “autoinducer-2” or AI-2”. The terms “autoinducer-2” and “AI-2” refer specifically to the signaling factor as produced by Vibrio harveyi. The terms “signaling factor” or “signaling compound”, “autoinducer” or “AI-2-like compound” refer generally to the signaling factors of the present invention, of which AI-2 is an example.
The term “isolated nucleic acid”, when applied to DNA, refers to a DNA that is separated from sequences with which it is immediately contiguous (in the 5′ and 3′ directions) in the naturally occurring genome of the organism from which it was derived. For example, the “isolated nucleic acid” may comprise a DNA inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a procaryote or eucaryote. An “isolated nucleic acid” may also comprise a cDNA.
The term “isolated nucleic acid”, when applied to RNA, refers to an RNA encoded by an isolated DNA as defined above. Alternatively, the term may refer to an RNA that has been sufficiently separated from RNAs with which it would be associated in its natural state (i.e., in cells or tissues), such that it exists in a “substantially pure” form (the term “substantially pure” is defined below).
The term “isolated protein” or “isolated and purified protein” refers primarily to a protein produced by expression of an isolated nucleic acid of the invention. Alternatively, this term may refer to a protein that has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in “substantially pure” form.
The term “substantially pure” refers to a preparation comprising at least 50–60% by weight the factor of interest (e.g., pathogenesis signaling factor, nucleic acid, oligonucleotide, protein, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90–99% by weight, the factor of interest. Purity is measured by methods appropriate for the factor of interest (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like).
With respect to antibodies of the invention, the term “immunologically specific” refers to antibodies that bind to one or more epitopes of a protein of interest, but that do not substantially recognize and bind other compounds in a sample containing a mixed population of antigenic biological constituents.
With respect to oligonucleotides, the term “specifically hybridizing” refers to the association between two single-stranded nucleotides of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed “substantially complementary”). In particular, the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of noncomplementary sequence.
The term “promoter region” refers to the transcriptional regulatory regions of a gene, which may be found at the 5′ or 3′ side of the coding region, or within the coding region, or within introns.
The term “selectable marker gene” refers to a gene encoding a product that, when expressed, confers a selectable phenotype such as antibiotic resistance on a transformed cell.
The term “reporter gene” refers to a gene that encodes a product that is easily detectable by standard methods, either directly or indirectly.
The term “operably linked” means that the regulatory sequences necessary for expression of the coding sequence are placed in the DNA in the appropriate positions relative to the coding sequence so as to enable expression of the coding sequence. This same definition is sometimes applied to the arrangement of transcription units and other regulatory elements (e.g., enhancers or translation regulatory sequences) in an expression vector.
The term “wild type cell” or “wild type strain” is used herein to describe cells or strains that serve as a reference point for cells or strains in which the expression level of a particular protein has been altered (i.e increased or decreased). Generally, the “wild type cell” or “wild type strain” and the cell or strain to which it is being compared will have the same genotype except for one or more difference that change the expression level of the protein. Thus, as used herein “wild type cells” or “wild type strains” may contain certain mutations that are shared with the cells or strains to which they are being compared but they do not share the genotype that confers altered expression levels of the protein of interest. For example, if a strain or cell expresses a higher level of a transporter than a wild type strain or cell, it may be genetically identical to the wild type strain or cell except for one or more mutations that are responsible for the increased expression level of the transporter.